Dna encoding novel enzyme having d-serine synthase activity, method of producing the enzyme and method of producing d-serine by using the same

ABSTRACT

This invention relates to DNA encoding a novel enzyme having activity of synthesizing D-serine from formaldehyde and glycine, recombinant DNA constructed by integrating such DNA into a vector, a transformant transformed with the recombinant DNA, and a method for producing D-serine from formaldehyde and glycine with the use of the enzyme.

This application is a Divisional Application of U.S. patent applicationSer. No. 11/665,194, filed Apr. 12, 2007, now U.S. Pat. No. 7,919,285,which is a National Stage Application of PCT/JP2005/018906, filed Oct.7, 2005, which claims priority to Japan 2004-298344 filed Oct. 13, 2004,all of which are incorporated by reference herein.

TECHNICAL FIELD

The present invention relates to DNA encoding a novel enzyme havingactivity of synthesizing D-serine from formaldehyde and glycine,recombinant DNA constructed by integrating such DNA into a vector, atransformant transformed with the recombinant DNA, a novel enzyme havingactivity of synthesizing D-serine from formaldehyde and glycine, and amethod for producing D-serine from formaldehyde and glycine with the useof the enzyme.

BACKGROUND ART

D-serine has been known as a compound that is useful as a synthesisintermediate for medicaments such as D-cycloserine.

Hitherto, as an enzyme having activity of synthesizing D-serine fromformaldehyde and glycine, only D-threonine aldolase (hereafter to beabbreviated as “DTA”), which is derived from Arthrobacter sp. DK-19, hasbeen known (JP Patent Publication (Kokai) No. 58-116690 A (1983)).

With the use of such DTA, it has been reported that the amount ofD-serine produced was 2.5 mmol when 50 mmol each of formaldehyde andglycine were subjected to reaction at 30° C. for 40 hours. (The yieldrelative to formaldehyde was merely 5%.)

Meanwhile, it has been reported that DTA derived from Arthrobacter sp.DK-38, which is a microorganism of the same genus Arthrobacter, has abroad substrate specificity that responds to D-threonine,D-β-hydroxyphenylserine, D-β-hydroxy-α-aminovaleric acid, and the like;however, it does not react with D-serine (see Eur. J. Biochem., 1997,248, pp. 385-393).

In addition, it has been reported that D-β-hydroxyamino acids can beproduced from glycine and an aldehyde compound with the use of DTAderived from the genus Xanthomonas (JP Patent Publication (Kokai) No.5-168484 A (1993)). However, it has not been reported that D-serine canbe synthesized from formaldehyde and glycine with the use of DTA derivedfrom the genus Xanthomonas.

DISCLOSURE OF THE INVENTION

In view of the aforementioned background art, it is an objective of thepresent invention to provide DNA encoding a novel enzyme having activityof synthesizing D-serine from formaldehyde and glycine, a recombinantDNA constructed by integrating such DNA into a vector, a transformanttransformed with the recombinant DNA, a novel enzyme having an activityof synthesizing D-serine from formaldehyde and glycine, and a method forproducing D-serine from formaldehyde and glycine with the use of theenzyme.

The present inventors thought that some unknown enzymes that havestructures similar to those of known DTAs could be capable ofsynthesizing D-serine from formaldehyde and glycine. Thus, they examinedinformation regarding the amino acid sequences of known DTAs.Accordingly, they have found information regarding the amino acidsequences of DTAs derived from the genera Xanthomonas (GenBank accessionNo. E05055), Achromobacter (GenBank accession No. AB026892), andArthrobacter (GenBank accession No. AB010956). As a result of comparisonand examination of these amino acid sequences, they have found thatamino acid sequences corresponding to the N-terminal region and theC-terminal region of DTA are highly homologous to each other.

The present inventors designed primers based on these amino acidsequences of the N-terminal region and the C-terminal region. They thentried to amplify chromosome DNAs of a variety of microorganisms by PCRusing the primers. Accordingly, they have succeeded in amplifying DNAencoding an enzyme having activity of synthesizing D-serine fromformaldehyde and glycine with the use of a microorganism of the genusAchromobacter.

The amino acid sequence corresponding to the DNA is approximately 50%homologous to the amino acid sequence of known DTA derived from thegenus Achromobacter, such that it significantly differs from the aminoacid sequence of known DTA derived from the genus Achromobacter.Meanwhile, it is approximately 90% homologous to the amino acid sequenceof known DTA derived from the genus Xanthomonas.

Next, the present inventors tried to synthesize D-serine by allowingformaldehyde to react with glycine using recombinant Escherichia colithat had been transformed with a recombinant DNA constructed byintegrating the above novel DNA into a vector. Surprisingly, unlike thecase of conventionally known DTA, they have found that D-serine can besynthesized with a reaction yield of as high as 70% or more from 100 mMglycine and 100 mM formaldehyde.

Further, when a D-serine accumulation reaction was carried out using therecombinant Escherichia coli, a small amount of L-serine was found to beproduced.

When D-serine is used in a field in which it is necessary for medicineintermediates or the like to have high purity, it is not preferable thatL-serine be mixed in D-serine. Formation of L-serine as a byproduct uponproduction of D-serine causes a significantly lowered purification yieldof D-serine, since the solubility of DL-serine is lower than that ofD-serine.

As a result of further examination to solve the above problems, thepresent inventors have found that formation of L-serine as a byproductcan be restrained by carrying out organic solvent treatment and/or heattreatment in the presence of divalent metal ions. In addition, they havefound that, even without carrying out such treatment, formation ofL-serine as a byproduct can be restrained by maintaining formaldehydeconcentration at 150 mM or more during reaction. Furthermore, they havefound that formation of L-serine as a byproduct can be restrained byusing a microorganism lacking an L-serine synthase gene as a hostproducing DSA that is used for D-serine synthesis.

Based on the above findings, the present invention has been completed.Specifically, the present invention is as follows.

(1) DNA encoding a protein described in the following (a) or (b):

(a) a protein comprising an amino acid sequence set forth in SEQ ID NO:4, 6, or 8; or

(b) a protein comprising an amino acid sequence derived from the aminoacid sequence of (a) by deletion, substitution, insertion, or additionof one to several amino acid residues and having enzyme activity ofsynthesizing D-serine from glycine and formaldehyde.

(2) DNA described in the following (a) or (b):

(a) DNA comprising a nucleotide sequence set forth in SEQ ID NO: 3, 5,or 7 in the Sequence Listing or a sequence complementary to thenucleotide sequence; or

(b) DNA hybridizing under stringent conditions with a DNA fragmentcomprising a DNA sequence comprising at least 20 consecutive bases ofthe nucleotide sequence set forth in SEQ ID NO: 3, 5, or 7 in theSequence Listing or of a sequence complementary to the nucleotidesequence and encoding an enzyme having an activity of synthesizingD-serine from glycine and formaldehyde.

(3) A recombinant DNA constructed by integrating the DNA described in(1) or (2) above into a vector.

(4) A transformant obtained by transforming a host cell using therecombinant DNA described in (3) above.

(5) The transformant described in (4) above, wherein the host cell to betransformed is a microorganism.

(6) The transformant described in (5) above, wherein the microorganismto be transformed is a D-serine-deaminase-deficient microorganism.

(7) A protein described in the following (a) or (b):

(a) a protein comprising an amino acid sequence set forth in SEQ ID NO:4, 6, or 8; or

(b) a protein comprising an amino acid sequence derived from the aminoacid sequence of (a) by deletion, substitution, insertion, or additionof one to several amino acid residues and having an enzyme activity ofsynthesizing D-serine from glycine and formaldehyde.

(8) A method for producing an enzyme having activity of synthesizingD-serine from glycine and formaldehyde, wherein the transformantdescribed in any one of (4) to (6) above is cultured such that a proteinhaving enzyme activity of synthesizing D-serine from glycine andformaldehyde is collected from the obtained culture product.

(9) A method for producing D-serine, comprising allowing glycine toreact with formaldehyde in the presence of the transformant described inany one of (4) to (6) above or a treated product thereof.

(10) A method for producing D-serine, comprising allowing glycine toreact with formaldehyde in the presence of the protein described in (7)above.

(11) A method for producing D-serine, wherein D-serine is synthesized byallowing glycine to react with formaldehyde in the presence of amicroorganism having activity of synthesizing D-serine from glycine andformaldehyde or a treated product thereof, comprising one or more meansdescribed in the following (i) to (iv) whereby formation of L-serine asa byproduct in a reaction solution is restrained in a manner such thatL-serine accounts for 1.5 mol% or less relative to D-serine during thereaction:

(i) a method for allowing a microorganism having activity ofsynthesizing D-serine from glycine and formaldehyde to be subjected toan organic solvent treatment and/or heat treatment;

(ii) a method for controlling formaldehyde concentration in a reactionsolution a) to 2M or less in a case in which an organic solventtreatment and/or heat treatment are/is carried out by the method (i)above and b) to from 150 mM to 2M inclusive in a case in which anorganic solvent treatment and/or heat treatment are/is not carried out;

(iii) a method for using, as a catalyst, a microorganism comprising anenzyme having activity of synthesizing D-serine from glycine andformaldehyde and lacking a L-serine synthase gene; and

(iv) a method for adding a microorganism comprising an enzyme having anL-serine deaminase activity to a reaction solution.

(12) The production method described in (11) above, wherein the organicsolvent is at least one selected from the group consisting offormaldehyde, benzaldehyde, dichloroethane, and isopropyl alcohol.

(13) The production method described in (11) or (12) above, wherein theorganic solvent treatment and/or heat treatment are/is carried out inthe presence of divalent metal ions.

(14) The production method described in (13) above, wherein the divalentmetals are one or more types of metals selected from the groupconsisting of magnesium, manganese, zinc, nickel, cobalt, and iron.

(15) The production method described in (11) above, wherein themicroorganism having activity of synthesizing D-serine from glycine andformaldehyde is the transformant described in (5) or (6) above.

1. Obtaining a Gene of DNA Encoding a Novel Enzyme Having Activity ofSynthesizing D-Serine from Formaldehyde and Glycine

DNA encoding the amino acid sequence set forth in SEQ ID NO: 4, 6, or 8in the Sequence Listing and encoding an enzyme having activity ofsynthesizing D-serine from glycine and formaldehyde (hereafter to beabbreviated as “DSA”) can be obtained by extracting chromosomal DNAfrom, for example, Achromobacter xylosoxidans (ATCC9220), which isavailable from the American Type Culture Collection (12301 ParklawnDrive, Rockville, Md. 20852, U.S.A.), and Achromobacter xylosoxidans(NBRC13495) and Achromobacter denitrificans (Synonym: Achromobacterxylosoxidans subsp. denitrificans) (NBRC15125), which are available fromthe NITE Biological Resource Center of the National Institute ofTechnology and Evaluation (2-5-8 Kazusakamatari, Kisarazu-shi, Chiba,Japan) and carrying out PCR with the use of primers comprising thenucleotide sequences set forth in SEQ ID NOS: 1 and 2 in the SequenceListing, respectively.

It is effective to improve amplification conditions in a manner suchthat a gene having a few mismatched bases can be amplified when carryingout PCR. For instance, a gene that is amplified with difficulty can beoccasionally amplified by a method wherein the annealing temperature iscontrolled at a low temperature, by a method involving the addition ofdimethyl sulfoxide (approximately 5%) to a PCR reaction solution, by amethod using a PCR kit (e.g., GC-RICH PCR System produced by Roche) thatis designed to readily amplify a GC-rich gene, or by a combination ofthe above methods or the like.

Specific examples of DNA encoding DSA include the DNA nucleotidesequence of a DTA gene derived from Achromobacter xylosoxidans(ATCC9220) set forth in SEQ ID NO: 3, the amino acid sequence(translated by such nucleotide sequence) set forth in SEQ ID NO: 4, theDNA nucleotide sequence of a DTA gene derived from Achromobacterxylosoxidans (NBRC13495) set forth in SEQ ID NO: 5, the amino acidsequence (translated by such nucleotide sequence) set forth in SEQ IDNO: 6, the DNA nucleotide sequence of a DTA gene derived fromAchromobacter denitrificans (NBRC15125) set forth in SEQ ID NO: 7, andthe amino acid sequence (translated by such nucleotide sequence) setforth in SEQ ID NO: 8.

In addition to the aforementioned DNAs encoding DSA, DNA encoding DSAmay be derived from any types of organisms, as long as it hybridizeswith a DNA fragment comprising a DNA sequence of at least 20 consecutivebases of the nucleotide sequence set forth in SEQ ID NO: 3, 5, or 7 inthe Sequence Listing or of a sequence complementary to such nucleotidesequence under stringent conditions and it encodes a protein having DSAactivity. For instance, even a silent DNA that does not function in anorganism may be used as long as it can produce DSA by ligating such DNAthat has been isolated to an adequate expression vector.

Further, even if no organism is specified, DNA encoding DSA can beobtained by allowing soil or the like that is used for template DNA tobe directly subjected to PCR using the primers having the nucleotidesequences set forth in SEQ ID NOS: 1 and 2 in the Sequence Listing.

A specific example of DNA encoding DSA is obtained by a method whereinPCR is carried out using, for example, chromosome DNA of a microorganismthat grows in a medium containing D-serine as a template and primershaving the nucleotide sequences set forth in SEQ ID NOS: 1 and 2 in theSequence Listing, such that DNA is amplified.

In addition, it is also possible to obtain DNA encoding a protein havingDSA activity and comprising an amino acid sequence derived from theamino acid sequence set forth in SEQ ID NO: 4, 6, or 8 by deletion,substitution, insertion, or addition of one to several amino acidresidues via introduction of adequate mutation such as deletion,substitution, insertion, and/or addition with the use of site-specificmutagenesis methods (Nucleic Acid Res., 10, p. 6487 (1982); Nucleic AcidRes., 13, p. 4431 (1985); Methods in Enzymol., 100, p. 448 (1983);Molecular Cloning 2^(nd) Ed., Cold Spring Harbor Laboratory Press(1989); PCR A Practical Approach IRL Press p. 200 (1991); CurrentProtocols in Molecular Biology, John Wiley & Sons (1987-1997); Proc.Natl. Acad. Sci., USA, 79, p. 6409 (1982); Gene, 34, 315 (1985); andProc. Natl. Acad. Sci., USA, 82, p. 488 (1985)) or the like, as long asthe nucleotide sequence set forth in SEQ ID NO: 3, 5, or 7 in theSequence Listing or a sequence complementary thereto can hybridize understringent conditions and such mutation does not influence the activityof the enzyme to be encoded.

Deletion, substitution, insertion, or addition of amino acid residuesdescribed herein can be carried out by the aforementioned site-specificmutagenesis methods that were known techniques prior to the filing ofthe present application. In addition, the term “one to several aminoacid residues” indicates a number of amino acids that allows deletion,substitution, insertion, or addition of amino acid residues (forexample, 1 to 5 amino acid residues and preferably 1 to 3 amino acidresidues) to be carried out by site-specific mutagenesis methods.

Examples of conditions for hybridization include conditions that aregenerally used by persons skilled in the art to detect particularhybridization signals. Preferably, such conditions indicate stringenthybridization conditions and stringent washing conditions. Specifically,for instance, such conditions involve overnight incubation at 55° C.with the use of a probe in a solution containing 6×SSC (1×SSCcomposition: 0.15 M NaCl and 0.015 M sodium citrate (pH 7.0)), 0.5% SDS,5× Denhardt's solution, and 100 mg/ml herring sperm DNA. Such conditionsalso involve washing of a filter in 0.2×SSC at 42° C. Stringentconditions involve a use of 0.1×SSC at 50° C. during a step of washing afilter. As a further stringent condition, a condition using 0.1×SSC at65° C. during the same step can be explained.

The phrase “a DNA fragment comprising a DNA sequence comprising at least20 consecutive bases of the nucleotide sequence set forth in SEQ ID NO:3, 5, or 7 or of a sequence complementary to such nucleotide sequence”used herein indicates a DNA fragment obtained by choosing one or moresequence comprising at least 20 and preferably at least 30 (e.g., 40,60, or 100) arbitrary consecutive bases of the sequence set forth in SEQID NO: 3, 5, or 7 in the Sequence Listing or of a sequence complementaryto such sequence.

2. Production of a Recombinant DNA Having the Gene of the Protein

A recombinant DNA can be obtained by integrating the aforementioned DNAencoding DSA into a vector. A vector that is appropriate for cloning isa vector that is constructed for gene recombination with the use of aphage or plasmid that is capable of autonomously replicating in a hostmicroorganism. For instance, when Escherichia coli is a hostmicroorganism, examples of such phage include Lambda gt10 and Lambdagt11. In addition, for instance, when Escherichia coli is a hostmicroorganism, examples of such plasmid include pBTrp2, pBTac1, andpBTac2 (produced by Boehringer Mannheim), pKK233-2 (produced byPharmacia), pSE280 (produced by Invitrogen), pGEMEX-1 (produced byPromega), pQE-8 (produced by QIAGEN), pQE-30 (produced by QIAGEN),pBluescriptII SK+ and pBluescriptII SK(−) (produced by Stratagene),pET-3 (produced by Novagen), pUC18 (produced by Takara Shuzo Co., Ltd.),pSTV28 (produced by Takara Shuzo Co., Ltd.), pSTV29 (produced by TakaraShuzo Co., Ltd.), and pUC118 (produced by Takara Shuzo Co., Ltd.).

As such promoter, any promoter may be used as long as it can beexpressed in a host cell. Examples thereof include promoters derivedfrom Escherichia coli, phages, or the like, such as a trp promoter(P_(trp)), a lac promoter (P_(lac)), a P_(L) promoter, a P_(H) promoter,and a P_(SE) promoter. In addition, promoters and the like that areartificially designed and modified, such as a tac promoter and a lacT7promoter, can be used. Further, in order to cause expression in bacteriaof the genus Bacillus, it is also possible to use an Np promoter (JPPatent Publication (Kokoku) No. 8-24586 B (1996)).

As a ribosome binding sequence, any sequence can be used as long as itcan be expressed in a host cell. However, it is preferable to use aplasmid in which a Shine-Dalgarno sequence and an initiation codon areadjusted to have an adequate distance (e.g., 6 to 18 bases)therebetween.

In order to efficiently carry out transcription and translation, aprotein, in which the N-terminal of a protein having activity of theprotein or a protein derived from such a protein by deletion of aportion thereof is fused with the N-terminal of a protein encoded by anexpression vector, may be expressed.

A transcription termination sequence is not always necessary for theexpression of a protein of interest. However, it is preferable to placea transcription termination sequence directly below a structural gene.

Upon cloning, it is possible to obtain a vector fragment by cleaving avector as described above with a restriction enzyme used for cleavage ofthe aforementioned DNA encoding DSA. However, the same restrictionenzyme used for cleavage of the DNA is not necessarily used. A methodfor binding the DNA fragment and a vector DNA fragment may be used aslong as a known DNA ligase is used in such a method. For instance, afterannealing of a cohesive end of the DNA fragment and that of a vectorfragment, a recombinant vector of the DNA fragment and the vector DNAfragment is produced with the use of an adequate DNA ligase. Also, it ispossible to produce a recombinant vector by transferring the resultantof such annealing into a host such as a microorganism and using an invivo DNA ligase according to need.

3. Production of a Transformant Using a Recombinant DNA Constructed byIntegrating DNA Encoding DSA into a Vector

A transformant transformed with the aforementioned recombinant DNA canbe obtained by introducing the aforementioned recombinant DNA into ahost cell.

As a host cell, there is no particular limitation, as long as arecombinant vector is stable and can autonomously replicate and aforeign gene is phenotypically expressed therein. Examples of such hostcell include microorganisms such as bacteria, including Escherichia colisuch as Escherichia coli DH5α and Escherichia coli XL-1Blue. Inaddition, it is possible to use other microorganisms such as yeasts orinsect cells as host cells.

In a case in which a microorganism is Escherichia coli, for example, themethod for transferring recombinant DNA into the microorganism that canbe used is a competent cell method using calcium treatment, anelectroporation method, or the like.

As a host cell, for the purpose of restraining degradation of D-serineas a product, it is possible to use a microorganism having low D-serinedeaminase activity or a microorganism lacking D-serine deaminaseactivity. Specific examples of a microorganism lacking D-serinedeaminase activity include Escherichia coli having a recombinantD-serine deaminase gene described in Example 6.

In addition, for the purpose of restraining production of L-serineduring a D-serine synthesis reaction, it is possible to degrade L-serineproduced using a microorganism having high L-serine deaminase activity.As a microorganism having high L-serine deaminase activity, Escherichiacoli having a recombinant L-serine deaminase gene described in Example15 or the like can be used.

As such microorganism, a microorganism having low activity of an enzymeinvolved in L-serine synthesis, such as alanine racemase, serinehydroxymethyltransferase, or L-threonine aldolase, or a microorganismlacking such enzyme activity is preferably used so that L-serine is notproduced.

Further preferably, a microorganism lacking all of the above enzymes isused as a host cell.

4. Production of DSA

DSA can be produced by culturing the aforementioned transformant andcollecting the thus obtained DSA.

A transformant can be cultured in accordance with a usual method usedfor culture of host cells. In a case in which a transformant is aprocaryotic microorganism such as Escherichia coli or a eucaryoticmicroorganism such as a yeast, a medium in which such a microorganism iscultured may be a natural or synthetic medium as long as it containscarbon sources, nitrogen sources, inorganic salts, and the like, whichcause assimilation of the microorganism, and as long as a transformantcan efficiently be cultured therein.

Examples of carbon sources that can be used include: glucose, fructose,or sucrose; molasses containing any thereof; carbohydrates such asstarch and starch hydrolysate; organic acids such as acetic acid andpropionic acid; and alcohols such as ethanol and propanol, as long asthey can cause assimilation of a transformant.

Examples of nitrogen sources that can be used include: ammonia; avariety of ammonium salst of inorganic or organic acid, such as ammoniumchloride, ammonium sulfate, ammonium acetate, and ammonium phosphate;other nitrogen-containing compounds; peptone; meat extracts; yeastextracts; corn steep liquor; casein hydrolysate; soybean cake; soybeancake hydrolysate; and a variety of fermentation bacteria and digeststhereof.

Examples of inorganic salts that can be used include monopotassiumphosphate, dipotassium phosphate, magnesium phosphate, magnesiumsulfate, sodium chloride, ferrous sulfate, manganese sulfate, coppersulfate, and calcium carbonate.

Culture is carried out under aerobic conditions used for shake culture,submerged cultivation under aeration and agitation, or the like. Culturetemperature is preferably 15° C. to 50° C. Culture time is 16 hours to 5days, in general. During culture, pH is maintained between 3.0 and 9.0.The pH is adjusted with the use of inorganic or organic acids, alkalinesolutions, urea, calcium carbonate, ammonia, and the like. In addition,during culture, antibiotics such as ampicillin and tetracycline may beadded to the medium according to need.

When a microorganism transformed with an expression vector in which aninducible promoter is used as a promoter is cultured, an inducer may beadded to the medium according to need. For instance, when amicroorganism transformed with an expression vector in which a lacpromoter is used is cultured, isopropyl-β-D-thiogalactopyranoside (IPTG)or the like may be added to the medium. Also, when a microorganismtransformed with an expression vector in which a trp promoter is used iscultured, indoleacetic acid (IAA) or the like may be added to themedium.

A transformant can be separated and recovered by means ofcentrifugation, filtration, or the like, of a culture solutioncontaining the transformant.

A treated product of a transformant can be obtained by allowing thetransformant to be subjected to mechanical disruption, ultrasonication,freezing and thawing treatment, drying treatment, pressurization ordepressurization treatment, osmotic pressure treatment, autodigestion,surfactant treatment, or enzyme treatment for the purpose of celldisruption. Also, a treated product of a transformant can be obtained asan immobilized fraction or transformant, which comprises DSA obtainedvia such treatment.

In addition, the treated product of a microorganism of the presentinvention is also referred to as a product subjected to a treatmentsimilar to that used for the aforementioned treated product of atransformant.

In order to purify DSA from such transformant or a treated product ofsuch transformant, transformed cells are disrupted and a cell disruptionsolution is subjected to a combination of, for example, fractionationvia ion-exchange resin or gel-filtration chromatography or the like andsalting out with the use of ammonium sulfate or the like, such that apurified enzyme can be obtained.

5. Production of D-Serine

D-serine can be produced by allowing glycine to react with formaldehydein the presence of the aforementioned transformant or a treated productof the transformant.

Production of D-serine is preferably carried out under conditions ofshaking or agitation at pH 6.0 to 9.0 and at a temperature of 20° C. to60° C.

The amount of transformant or treated product of the transformant usedis not particularly limited, as long as the reaction betweenformaldehyde and glycine progresses well. In general, a transformant ora treated product of the transformant is preferably added in an amountof at least 10 units and preferably 50 units or more in terms of DSAactivity relative to 1 g of glycine. In accordance with a method foradding the transformant or a treated product of the transformant, suchtransformant may be added at once upon the initiation of reaction, atseveral different times, or continuously throughout reaction.

Regarding DSA activity, capacity for synthesizing 1 μmol of D-serine per1 minute is defined as 1 unit.

DSA activity is calculated by measuring the amount of D-serine that isproduced in a manner such that an enzyme solution is added to 200 mMTris-hydrochloric acid buffer (pH 8.0) containing 100 mM glycine, 0.1 mMpyridoxal phosphate, 10 mM magnesium chloride, and 5 mM formaldehyde,followed by incubation at 30° C.

Glycine concentration in a reaction solution is 100 mM or more andpreferably between 1 M and 5 M. In accordance with a method for addingglycine, it may be added at once upon the initiation of reaction, atseveral different times, or continuously along with the progress ofreaction.

It is possible to supply formaldehyde in the form of gas into a reactionsolution. Also, it can be supplied in the form of an aqueous or alcoholsolution. In addition, as a supply source of formaldehyde,paraformaldehyde can also be used. However, an aqueous solution ofapproximately 37% formaldehyde is preferably used.

As a method for adding formaldehyde, a method of adding formaldehyde atonce or a method of adding formaldehyde at several different times orcontinuously along with the progress of reaction can be carried out.Preferably, formaldehyde concentration in a reaction solution iscontrolled at a concentration that allows DSA activity not to beinhibited. A concentration that allows DSA activity not to be inhibitedis generally 5 M or less, preferably 2 M or less, further preferably 500mM or less, and particularly preferably 300 mM or less.

Also, formaldehyde can be added by the following methods for controllingformaldehyde concentration in a reaction solution: (1) a method foradding formaldehyde in a reaction solution at a given rate; (2) a methodwherein formaldehyde concentration is quantified such that formaldehydeis added at several different times at a concentration that allowsenzyme activity not to be deactivated; (3) a method for substantiallyavoiding inhibition due to formaldehyde, wherein paraformaldehyde isadded and an enzyme is added to a reaction system at a rate that exceedsthe rate at which formaldehyde is released from paraformaldehyde; and(4) a method for adding formaldehyde in an amount at which an increasedpH level can be corrected. In the case of (4) above, D-serine as aproduct accumulates in a reaction solution along with the progress ofreaction, the amount of glycine as a starting material decreases, andthe pH of a reaction solution decreases along with the progress of thereaction. This is because the isoelectric point of D-serine is 5.68 andthat of glycine is 5.97, such that alkali is added to the reactionsolution in an amount that exceeds an amount necessary for correction ofa decrease in the pH of the reaction solution, resulting in an increasedpH level.

When formaldehyde is added at a rate that exceeds the rate of addingalkali while the amount of DSA in a reaction solution is predetermined(that is to say, at a rate that exceeds the rate of D-serine synthesis),it becomes possible to control formaldehyde concentration in a reactionsolution without using complicated operations such as measurement offormaldehyde concentration.

Examples of alkali that may be added to a reaction solution include:alkali metal hydroxide such as lithium hydroxide, sodium hydroxide, orpotassium hydroxide; ammonium hydroxide; calcium hydroxide; dipotassiumphosphate; disodium phosphate; potassium pyrophosphate; and ammonia, aslong as it is dissolved in water such that basicity is imparted to theliquid.

A medium used for a reaction solution is water, an aqueous medium, anorganic solvent, or a mixture solution of water or an aqueous medium andan organic solvent. Examples of an aqueous medium that is used includebuffer solutions such as a phosphate buffer solution, a HEPES(N-2-hydroxyethylpiperazin-N-ethanesulfonic acid) buffer solution, and aTris(Tris(hydroxymethyl)aminomethane)hydrochloric acid buffer solution.Examples of an organic solvent that may be used include any organicsolvent such as acetone, ethyl acetate, dimethyl sulfoxide, xylene,methanol, ethanol, or butanol, unless it inhibits reaction.

In the aforementioned reaction, reaction yield may be improved with theaddition of a compound having divalent metal ions, reductants such as2-mercaptoethanol, dithiothreitol, and sodium bisulfite, and coenzymessuch as pyridoxal phosphate and an ammonium salt.

Examples of a compound having divalent metal ions include magnesiumchloride, manganese chloride, cobalt acetate, ferrous sulfate, andcalcium chloride.

The concentration of such compound in a reaction solution is generally0.1 mM to 100 mM and preferably 0.1 mM to 10 mM.

6. Method for Improving Optical Purity of D-Serine in a ReactionSolution

Formation of L-serine as a byproduct can be restrained by allowingglycine to react with formaldehyde in the presence of a treated productobtained by allowing the aforementioned transformant or a treatedproduct of the transformant to be subjected to heat treatment or in thepresence of a treated product obtained by allowing the aforementionedtransformant or a treated product of the transformant to be subjected toorganic solvent treatment.

As conditions of heat treatment, any conditions may be applied, providedthat thereby DSA activity is not significantly reduced by heating andL-serin production activity can be reduced or eliminated. Specificexamples of such treatment include a method of agitation at pH 6.0 to9.0 at a temperature of 40° C. to 70° C. for 10 minutes to 6 hours.

In addition, organic solvent treatment and heat treatment can be used incombination.

As conditions of organic solvent treatment, any conditions may beapplied, provided that thereby DSA activity is not significantly reducedand L-serin production activity can be reduced or eliminated. Suchconditions of organic solvent treatment involve organic solventconcentration that is generally approximately between 20 mM and 2 M,preferably approximately between 20 mM and 1 M, further preferablyapproximately between 50 mM and 1000 mM, and particularly preferablyapproximately between 50 mM and 300 mM. Preferred examples of an organicsolvent that is used include: aldehydes such as formaldehyde,acetaldehyde, and benzaldehyde; alcohols such as methanol, ethanol, andisopropyl alcohol; ketones such as acetone; and halogenated hydrocarbonssuch as dichloroethane. However, the organic solvent is not limitedthereto unless it causes a significant decrease in DSA activity. Amongthem, formaldehyde is the most preferable because it is a substrate forenzyme reactions. Since the addition of D-serine results in productionof formaldehyde during such enzyme reaction, a method for addingD-serine instead of formaldehyde may be implemented. It is desired thatthe concentration of D-serine added be approximately 100 mM to 5 M.

It is desired that the temperature for organic solvent treatment bebetween 10° C. and 50° C. and that the pH for the treatment be between6.0 and 9.0. During organic solvent treatment, agitation is desirablycarried out such that the pH and the organic solvent concentrationbecome uniform in a treatment solution.

In addition, upon the initiation of reaction of D-serine production, itis possible to reduce or deactivate L-serine production activity byincreasing formaldehyde concentration. In such case, formaldehydeconcentration during reaction is maintained at approximately 0.1% to 5%for 30 minutes to 3 hours and then a general reaction may be carriedout.

When carrying out the aforementioned treatment, enzyme activity may befurther stabilized by adding a compound having divalent metal ions,reductants such as 2-mercaptoethanol, dithiothreitol, and sodiumbisulfite, and coenzymes such as pyridoxal phosphate and an ammoniumsalt.

Examples of a compound having divalent metal ions include magnesiumchloride, manganese chloride, cobalt acetate, ferrous sulfate, andcalcium chloride.

A concentration of such compound in a treatment solution is generally0.1 mM to 100 mM and preferably 0.1 mM to 10 mM.

It is also possible to improve optical purity of D-serine as a finalproduct by adding L-serine-deaminase-producing bacteria to a reactionsolution after the termination of a D-serine synthesis reaction so as todegrade the L-serine produced. As L-serine deaminase-producing bacteria,microorganisms lacking D-serine deaminase are desirably used.

7. Method for Collecting D-Serine

D-serine can be collected from a reaction solution in accordance withmethods that are used in general organic synthetic chemistry, such asextraction using organic solvents, crystallization, thin-layerchromatography, and high-performance liquid chromatography.

In accordance with the present invention, D-serine can be produced fromglycine and formaldehyde with a better yield than is possible withmethods for producing D-serine using known DTAs.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the results of SDS-polyacrylamide gel electrophoresisanalysis of a cell disruption solution obtained from transformants.

FIG. 2 shows an SDS-polyacrylamide gel electrophoresis image of purifiedDSA.

This description includes part or all of the contents as disclosed inthe description and/or drawings of Japanese Patent Application No.2004-298344, which is a priority document of the present application.

BEST MODE FOR CARRYING OUT THE INVENTION

Examples of the present invention will hereafter be described. However,the technical scope of the present invention is not limited to theExamples. In addition, D-serine, L-serine, and glycine were quantifiedby high-performance liquid chromatography. Conditions for analyzing themand a method for measuring activities of enzymes (DSA and DTA) were asfollows.

(1) Conditions for Analyzing D-Serine and L-Serine

Column: TSK-GEL ENANTIO L1 4.6×250 (Tosoh Corporation)

Column temperature: 45° C.

Pump flow rate: 0.8 ml/min.

Detection: UV 254 nm

Eluent: 0.25 mM copper sulfate: methanol=9:1 (v/v)

(2) Conditions for Analyzing Serine and Glycine

Column: Shodex RSpak NN-814 8×250 (Showa Denko K.K.)

Column temperature: 40° C.

Eluent: 10 mM potassium phosphate (pH 3.0)

Pump flow rate: 0.8 ml/min

Detection was carried out by a post-column derivatization method (J.Chromatogr., 83, 353-355 (1973)) using orthophthalaldehyde (OPA).

(3) A Method for Measuring Enzyme Activity

An enzyme solution that had been obtained by allowing a cell suspensionto be subjected to ultrasonic disruption was adequately diluted. Thediluted enzyme solution (0.1 mL) was added to 0.9 mL of 200 mMTris-hydrochloric acid buffer (pH 8.0) containing 100 mM glycine, 0.1 mMpyridoxal phosphate, 10 mM magnesium chloride, and 5 mM formaldehyde.The resulting solution was subjected to reaction at 30° C. for 15minutes.

D-serine produced was analyzed by HPLC so as to measure the activity.Herein, 1 unit of such activity was determined to be the capacity forproducing 1 μmol of D-serine per 1 minute.

EXAMPLE 1 Obtaining the Gene Encoding DSA

An LB medium (50 ml) was inoculated with Achromobacter xylosoxidans(ATCC9220), Achromobacter denitrificans (NBRC15125), and Achromobacterxylosoxidans (NBRC13495). After overnight culture at 30° C., harvest wascarried out, followed by bacteriolysis using a lytic solution containinglysozyme (1 mg/ml). The resulting lysate was subjected to phenoltreatment. Then, DNA was allowed to precipitate by ethanol precipitationin accordance with a usual method. The resulting DNA precipitate wasrecovered by spooling it onto a glass rod and washed so as to be usedfor PCR.

Primers used for PCR were oligonucleotides (obtained by custom synthesisfrom Hokkaido System Science Co., Ltd.) having the nucleotide sequencesset forth in SEQ ID NOS: 1 and 2, respectively, which were designedbased on known DTA genes. These primers had KpnI and HindIII restrictionenzyme recognition sequences near the 5′ and 3′ ends, respectively.

With the use of 0.025 ml of a PCR reaction solution containing 6 ng/μleach of chromosome DNAs of the aforementioned microorganisms and 3 μMeach of the primers, PCR was carried out under the following conditions:denaturation at 96° C. for 1 minute, annealing at 55° C. for 30 seconds,and elongation reaction at 68° C. for 1 minute and 15 seconds for 35reaction cycles.

The PCR reaction product and plasmid pUC18 (Takara Shuzo) were digestedwith KpnI and HindIII, followed by ligation using Ligation High(TOYOBO). Thereafter, the obtained recombinant plasmid was used fortransformation of Escherichia coli DH5α. The transformed cell line wascultured in an LB agar medium containing 50 μg/ml of ampicillin (Am) andX-Gal (5-bromo-4-chloro-3-indolyl-β-D-galactoside). Thus, anAm-resistant transformed cell line that was formed into a white colonywas obtained. A plasmid was extracted from the thus obtained transformedcell line. The nucleotide sequence of the DNA fragment that had beenintroduced into the plasmid was confirmed in accordance with a usualmethod for base sequencing.

Molecular weights of amino acid sequences that were expected based onthe obtained DNAs encoding DSA were approximately 40 kDa each.

The obtained plasmid having DNA encoding DSA derived from Achromobacterxylosoxidans (ATCC9220) was designated as pAcDTA1.

The plasmid having DNA encoding DSA derived from Achromobacterxylosoxidans (NBRC13495) was designated as pAcDTA2. The plasmid havingDNA encoding DSA derived from Achromobacter denitrificans (NBRC15125)was designated as pAcDTA3.

(Production of Transformants)

Escherichia coli DH5α was transformed by a usual method using pAcDTA1,pAcDTA2, and pAcDTA3. The obtained transformants were designated asMT-11015, MT-11016, and MT-11017, respectively.

A LB medium (100 mL) containing 50 μg/ml of Am was inoculated withrecombinant microorganisms of each transformant after being placed in a500-mL baffled Erlenmeyer flask. This was followed by culture at 30° C.until OD660 reached 0.6. Then, IPTG (isopropyl-β-thiogalactopyranoside)was added thereto such that the medium contained 1 mM IPTG. This wasfollowed by further shake culture for 16 hours. The culture solution wascentrifuged at 13000 rpm for 10 minutes. The obtained cell bodies weresuspended in 100 mM Tris-hydrochloric acid buffer (pH 8.0) containing 5mL of 1 mM magnesium chloride, followed by cryopreservation at −20° C.

EXAMPLE 2 Method for Producing DSA

Suspensions (0.5 mL each) of the transformants produced in Example 1were disrupted using a Bioruptor (produced by Olympus) in ice water for5 minutes. A transformant disruption solution was centrifuged such thatthe transformant disruption solution was prepared. FIG. 1 shows theresults of SDS-polyacrylamide gel electrophoresis analysis of thetransformant disruption solution.

0.5 mL of 100 mM Tris-hydrochloric acid buffer (pH 8.0) was added to theprecipitate so as to obtain cell residue, followed by analysis in asimilar manner.

In a soluble fraction of each transformant, a protein that was expressedat a position of 40 kDa was found. However, such protein was not foundin an insoluble fraction. The molecular weight of the protein was almostequivalent to the molecular weight of an amino acid sequence that wasbased on the corresponding gene.

EXAMPLE 3 Purification of DSA and D-Serine Synthesis with the Use ofPurified DSA

10 mL of a MT-11015 disruption solution produced in a manner similar tothat used in Example 2 was centrifuged at 10000 rpm for 20 minutes suchthat an enzyme solution was prepared, and cell residue was then removedtherefrom. The enzyme solution was allowed to adsorb anion exchangeresin (HiTrap Q-XL produced by Amersham), followed by linear-gradientelution from 100 mM Tris-hydrochloric acid buffer (pH 8.0) containing 10mM magnesium chloride and 50 mM sodium chloride to 100 mMTris-hydrochloric acid buffer (pH 8.0) containing 10 mM magnesiumchloride and 500 mM sodium chloride. The activity fraction was allowedto adsorb hydrophobic chromatography resin (HiTrap Phenyl FF produced byAmersham), followed by linear-gradient elution from 100 mMTris-hydrochloric acid buffer (pH 8.0) containing 10 mM magnesiumchloride, which had been saturated with ammonium sulfate, to 100 mMTris-hydrochloric acid buffer (pH 8.0) containing 10 mM magnesiumchloride. Note that the above operations were carried out atapproximately 10° C.

FIG. 2 shows the results of SDS-polyacrylamide gel electrophoresisanalysis of a solution subjected to ultrasonic disruption, an activityfraction subjected to ion-exchange chromatography treatment, and anactivity fraction subjected to hydrophobic chromatography treatment. Themolecular weight of purified DSA monomer was 40000±5000.

The purified DSA enzyme solution (150 units) was added to a substratesolution comprising 100 mM formaldehyde, 100 mM glycine, 0.1 mM PLP, 10mM magnesium chloride, and 100 mL of 200 mM Tris-hydrochloric acidbuffer (pH 8.0). The resultant was subjected to reaction at 30° C. for20 hours.

The reaction yield of D-serine was 95%.

EXAMPLE 4 Comparison Between D-Serine Synthesis Capacity and D-ThreonineSynthesis Capacity

The enzyme solution subjected to ultrasonic disruption (150 units) ofthe cell line MT-11015 produced in Example 1 was added to a substratesolution comprising 100 mM formaldehyde or acetaldehyde as an aldehydesource, 100 mM glycine, 0.1 mM PLP, 10 mM magnesium chloride, and 100 mLof 200 mM Tris-hydrochloric acid buffer (pH 8.0). The resultant wassubjected to reaction at 30° C. for 20 hours.

When formaldehyde was used as an aldehyde source, the yield was 90%.Also, when acetaldehyde was used as aldehyde source, the yield was 10%.

EXAMPLE 5 D-serine Synthesis Reaction at a Formaldehyde Concentration of100 mM

The enzyme solutions (150 units each) subjected to ultrasonic disruptionof the recombinant microorganisms produced in Example 1 were separatelyadded to a substrate solution comprising 100 mL of 200 mMTris-hydrochloric acid buffer (pH 8.0) containing 100 mM formaldehyde,100 mM glycine, 0.1 mM PLP, and 10 mM magnesium chloride. The resultantswere subjected to reaction at 30° C. for 20 hours. Table 1 shows theresults.

TABLE 1 Reaction Host Plasmid yield DH5α pAcDTA1 90% pAcDTA2 80% pAcDTA372%

EXAMPLE 6 Production of D-Serine Deaminase-Deficient Escherichia Coli

The entire nucleotide sequence (GenBanak accession number: U00096) ofgenomic DNA of Escherichia coli is known to the public. Also, the aminoacid sequence of Escherichia coli D-serine deaminase and the nucleotidesequence (GenBank accession number: J01603) of the gene thereof(hereafter to be abbreviated in some cases as dsdA) have already beenreported. PCR was carried out using genomic DNA of Escherichia coli cellline W3110 (ATCC27325) as a template and oligonucleotides having thenucleotide sequences set forth in SEQ ID NOS: 9, 10, 11, and 12, whichhad been produced based on genetic information regarding a region in thevicinity of dsdA of genomic DNA of Escherichia coli cell line W3110. Theobtained DNA fragments were digested with PstI and XbaI and with XbaIand KpnI, respectively, which are restriction enzymes. Thus,approximately 900-bp and 800-bp fragments of each DNA fragment wereobtained. The resulting DNA fragments were mixed with fragments obtainedby digesting a temperature-sensitive cloning vector pTH18cs1 (GenBankaccession number: AB019610) (Hashimoto-Gotoh, T., Gene, 241, 185-191(2000)) with PstI and KpnI, followed by ligation using a ligase. Theresultant was transformed into a DH5α cell line at 30° C. Thus, atransformant that was able to grow on an LB agar plate containing 10μg/ml of chloramphenicol was obtained. The obtained colony was culturedovernight at 30° C. in an LB liquid medium containing 10 μg/ml ofchloramphenicol so that a plasmid was recovered from the obtained cellbodies. The obtained plasmid was digested with XbaI so as to besubjected to blunt-end treatment with T4DNA polymerase. Thereafter, theplasmid was ligated with a kanamycin-resistant gene derived from pUC4Kplasmid (Pharmacia).

The thus obtained plasmid was transformed into Escherichia coli cellline W3110 (ATCC27325) at 30° C., followed by overnight culture at 30°C. on an LB agar plate containing 10 μg/ml of chloramphenicol and 50μg/ml of kanamycin. Thus, a transformant was obtained. An LB liquidmedium containing 50 μg/ml of kanamycin was inoculated with the obtainedtransformant, followed by overnight culture at 30° C. Next, theresultant was applied to an LB agar plate containing 50 μg/ml ofkanamycin so as to obtain the culture cell bodies. Thus, colonies thatwere able to grow at 42° C. were obtained. The obtained colonies werecultured overnight at 30° C. in an LB liquid medium containing 50 μg/mlof kanamycin. The resultant was further applied to an LB agar platecontaining 50 μg/ml of kanamycin so as to obtain colonies that were ableto grow at 42° C.

100 colonies were randomly picked up from the colonies that appeared.Each of them was allowed to grow on an LB agar plate containing 50 μg/mlof kanamycin and on an LB agar plate containing 10 μg/ml ofchloramphenicol. Then, chloramphenicol-sensitive clones that exclusivelygrow on an LB agar plate containing kanamycin were selected. Further,fragments (of approximately 3.0 kb) in the region in the vicinity ofdsdA were amplified by PCR from chromosome DNAs of these clones ofinterest. Then, a cell line in which dsdA had been substituted with akanamycin-resistant gene was selected. The obtained cell line wasdesignated as a W3110dsdA-deficient cell line (hereafter to beabbreviated in some cases as AdsdA). Transformation was carried outusing the plasmids produced in Example 1 so that cryopreservated cellbodies were produced as described above. Then, a similar reaction wascarried out. As a result, substantially no D-serine degradation wasconfirmed.

EXAMPLE 7 Production of D-Serine with the Use of a TransformantMT-11016: Production Without Addition of Mg

Distilled water (53.1 g) was added to 7.5 g of glycine and 9.4 g ofpyridoxal phosphate (0.026% by weight). The resultant was adjusted to pH8.0 with sodium hydroxide. A cell suspension (corresponding to 1500units in terms of activity) of MT-11016 obtained in Example 1 was addedthereto. Then, 20.8 g of formaldehyde (20% by weight) was added theretoat a reaction temperature of 30° C. in a manner such that formaldehydeconcentration in the reaction solution was quantified by the AHMT method(Eisei Kagaku (Journal of Health Science) (1976), Vol. 22, p. 39) asbeing between 50 mM and 300 mM. The pH of the reaction solution wasadjusted to pH 8.0 with sodium hydroxide. The reaction yield after 72hours reached 85%.

EXAMPLE 8 Production of D-Serine with the Use of a TransformantMT-11017: Production Without Addition of Mg

Distilled water (53.1 g) was added to 7.5 g of glycine and 9.4 g ofpyridoxal phosphate (0.026% by weight). The resultant was adjusted to pH8.0 with sodium hydroxide. A cell suspension (corresponding to 1500units in terms of activity) of MT-11017 obtained in Example 1 was addedthereto. Then, 20.8 g of formaldehyde (20% by weight) was added to thereaction solution at a reaction temperature of 30° C. by repeatingcycles of addition of formaldehyde at a rate of 0.8 g/15 minutes for 15minutes and discontinuation of the addition of formaldehyde for 45minutes. The pH of the reaction solution was adjusted to pH 8.0 withsodium hydroxide during reaction. After 24 hours, the reaction yield ofD-serine reached 95%.

Meanwhile, when formaldehyde was added at once so as to be subjected toreaction, the reaction yield was 20%.

EXAMPLE 9 Production of D-Serine with the Use of a Transformant Treatedwith Formaldehyde: Production Without Addition of Mg

Formaldehyde was added to a lysate of the frozen cell bodies of MT-11015produced in Example 1 such that the lysate contained 100 mMformaldehyde, followed by mild agitation at 30° C. for 1 hour. Duringagitation, the pH was adjusted to 8.0 with sodium hydroxide. Theresulting suspension of the cell bodies was subjected to the samereaction as that of Example 8. When formaldehyde treatment was notcarried out, 2 mol % of L-serine was produced. When formaldehydetreatment was carried out, it was impossible to detect L-serine.

6N-hydrochloric acid was added to the above reaction solution such thatthe solution was adjusted to pH 4.1. Activated carbon (0.97 g; watercontent of 50%) was added thereto, followed by agitation at 60° C. for 1hour. Activated carbon and cell body components were removed byfiltration. Then, the filtrate was concentrated to 30 g. Isopropylalcohol (13 g) was gradually added thereto, followed by mild agitationon ice for 1 hour. Thus, D-serine was allowed to deposit. The solutioncontaining crystal deposits was filtrated. The crystal deposits werewashed with 13 mL of cooled 40% isopropyl alcohol, followed bydehydration. The recovery rate was 60%, and white D-serine crystal wasobtained. Glycine as a starting material was not detected. The opticalpurity of the crystal was 99.8% ee.

EXAMPLE 10 [10-1] (Cloning of DTA of Xanthomonas and VectorConstruction)

An LB medium (50 ml) was inoculated with Xanthomonas oryzae (IAM1657),which is obtainable from the Institute of Molecular and CellularBiosciences at the University of Tokyo. After overnight culture at 30°C., harvest was carried out, followed by bacteriolysis using a lyticsolution containing lysozyme (1 mg/ml). The resulting lysate wassubjected to phenol treatment. Then, DNA was allowed to precipitate byethanol precipitation in accordance with a usual method. The resulting

DNA precipitate was recovered by spooling it onto a glass rod and washedso as to be used for PCR.

Primers used for PCR were oligonucleotides (obtained by custom synthesisfrom Hokkaido System Science Co., Ltd.) having the nucleotide sequencesset forth in SEQ ID NOS: 13 and 14, respectively, which were designedbased on a known DTA gene of Xanthomonas oryzae (GenBanak accessionnumber: E05055). These primers had KpnI and HindIII restriction enzymerecognition sequences near the 5′ and 3′ ends, respectively.

With the use of 0.025 ml of a PCR reaction solution containing 6 ng/μleach of chromosome DNAs of the aforementioned microorganisms and 3 μMeach of the aforementioned primers, PCR was carried out under thefollowing conditions: denaturation at 96° C. for 1 minute, annealing at55° C. for 30 seconds, and elongation reaction at 68° C. for 1 minuteand 15 seconds for 35 reaction cycles.

The PCR reaction product and plasmid pUC18 (Takara Shuzo) were digestedwith KpnI and HindIII, followed by ligation using Ligation High(TOYOBO). Thereafter, the obtained recombinant plasmid was used fortransformation of Escherichia coli DH5α. The transformed cell line wascultured in an LB agar medium containing 50 μg/ml of ampicillin (Am) andX-Gal (5-bromo-4-chloro-3-indolyl-β-D-galactoside). Thus, anAm-resistant transformed cell line that was formed into a white colonywas obtained. A plasmid was extracted from the thus obtained transformedcell line. The nucleotide sequence of the DNA fragment that had beenintroduced into the plasmid was confirmed to be a sequence identical tothe sequence of DTA of Xanthomonas oryzae in accordance with a usualmethod for base sequencing. The obtained expression plasmid wasdesignated as pXDTA1.

[10-2] (Obtaining Escherichia Coli Expressing DTA of the GenusXanthomonas)

Escherichia coli W3110ΔdsdA was transformed by a usual method usingpXDTA1. The obtained transformed cell line was designated as MT-11028.

In addition, Escherichia coli W3110ΔdsdA was transformed by a usualmethod using pAcDTA1, pAcDTA2, and pAcDTA3 obtained in Example 1. Theobtained transformants were designated as MT-11015W, MT-11016W, andMT-11017W, respectively.

[10-3] (Obtaining Cell Bodies Via Jar Culture)

A LB medium (100 mL) containing 50 μg/ml of Am was inoculated withrecombinant Escherichia coli (MT-11015W, MT-11016W, MT-11017W, andMT-11028) after being placed in a 500-mL baffled Erlenmeyer flask. Thiswas followed by culture at 30° C. until OD660 reached 1.0.

Subsequently, culture was carried out using BMS10 with capacity of 10 L(produced by ABLE). Operations were carried out under the followingculture conditions: agitation: 700 rpm; temperature: 30° C.; pH(maintained with NH₃): 7.2; aeration: 1 vvm; capacity: 5 L; and culturetime: 48 hours. A medium used had a medium composition comprising 7 g ofpolypeptone (Dainippon Pharma), 0.09 g of ferrous sulfate heptahydrate,1.5 g of ammonium sulfate, 2 g of magnesium sulfate hexahydrate, 2 g ofmonopotassium hydrogen phosphate, 2 g of dipotassium hydrogen phosphate,and 0.6 g of ADEKA NOL LG126 (Asahi Denka Kogyo K.K.) with respect to 1L of water, unless specified.

Before inoculation, glucose was added, resulting in a glucoseconcentration of 20 g/L. Then, 50 mL of the culture solution in theaforementioned baffled Erlenmeyer flask was used for inoculation. Afterthe glucose that had been added first became depleted under theaforementioned conditions, glucose was supplied at a variable rate (thatresulted in less than 0.1 g/L of glucose) during the remaining time suchthat the total amount of glucose added was 200 g. Cell bodies werecollected from the culture solution via centrifugation so as to befrozen at −20° C.

[10-4] (Restraining of Formation of L-Serine as a Byproduct from aMicroorganism Treated with an Organic Solvent)

[Method for Measuring Enzyme Activity of Formation of L-Serine as aByproduct]

Magnesium chloride hexahydrate (1.0 g) was added to 60 g of frozen cellbodies of MT-11028 (with a solid content percentage of approximately10%). A variety of organic solvents were added thereto in a manner suchthat the resultant contained the solvents at given concentrations,followed by agitation at 35° C. for 1 hour.

Cell bodies (weighing 0.22 g as dry cell bodies) were taken from theabove processed cell solution. The solution (9 g) used for enzymeactivity measurement was added thereto, followed by agitation at 35° C.for 20 hours. Then, the ratio between L-serine produced and residualD-serine was measured. Table 2 collectively shows the results.

(Solution Used for L-Serine Production Activity Measurement)

D-serine (10.84 g) and PLP (6 mg) were dissolved in 0.5 M potassiumphosphate buffer (pH 7.0) such that 100 g of the resultant was obtained.

TABLE 2 DSA Type of organic Concentration Optical purity residualsolvent upon treatment of D-serine activity Water   93%  95% Isopropylalcohol  20% by weight   96%  50% Benzaldehyde   5% by weight 97.5%  82%Dichloroethane   5% by weight 98.2% 103% Formaldehyde 0.5% by weight98.7%  46%

EXAMPLE 11 D-Serine Synthesis with the Use of Microorganisms Treatedwith an Organic Solvent

Magnesium chloride hexahydrate (1.85 g) and 1.2 g of formaldehyde (37%by weight) were added to 83.4 g of wet cell bodies (with a solid contentpercentage of approximately 10%) obtained in Example 10. Water was addedthereto such that the formaldehyde concentration was adjusted to 0.5%,followed by agitation at 35° C. for 1 hour.

Glycine (80 g), 4 g of magnesium chloride hexahydrate (35% by weight),and 3.1 g of formaldehyde (37% by weight) were added to 280 g of water.The pH of the resultant was adjusted to 7.5 with sodium hydroxide.

3.2 g of a PLP solution (0.38% by weight) was added thereto. Reactionwas initiated by adding 30 g of the above cell bodies treated with anorganic solvent as wet cell bodies. During reaction, formaldehyde wasadded when the pH became higher than 7.3 so that the pH was controlledat 7.3. The formaldehyde concentration during reaction was obtained bysubtracting the amount of D-serine produced that was quantified by HPLCfrom the amount of formaldehyde added. The formaldehyde concentration inthe reaction solution was controlled approximately between 80 mM and 100mM. As a result of analysis of serine after the termination of reactionby HPLC, the yield relative to that of Gly was 95 mol % and the opticalpurity was 99.9% ee.

EXAMPLE 12 Example of Reaction with High Formaldehyde Concentration

Glycine (80 g) and 4 g of magnesium chloride hexahydrate (35% by weight)were added to 280 g of water so as to be dissolved therein. Formaldehydewas added thereto such that the concentrations listed below wereachieved, followed by control of pH at 7.5 with the use of sodiumhydroxide. After the addition of 3.2 g of PLP (0.38% by weight), 30 g offrozen cell bodies obtained in Example 10 were added thereto so as toinitiate reaction. When the pH reached 7.3 or more during reaction,formaldehyde was added such that pH was controlled at 7.3. Table 3collectively shows the results.

TABLE 3 Formaldehyde Rea- Micro- concentration in reaction Optical ctionorganism solution purity yield Comparative MT-11028 10 to 20 mM 96.5%77.9% example Examples MT-11028 Approximately 150 mM 98.1% 98.0%Approximately 330 mM 99.8% 98.5% Approximately 410 mM 99.9% 96.6%Approximately 660 mM 99.9% 95.0% Approximately 1300 mM 99.9% 95.0%Approximately 2000 mM 99.9% 90.0% MT-11015W Approximately 410 mM 99.9%89.7% MT-11016W Approximately 410 mM 99.8% 96.6% MT-11017W Approximately410 mM 99.8% 90.5%

EXAMPLE 13

[13-1] (Construction of Escherichia Coli in which the GlyA Gene isDestroyed and Production of DSA-Producing Bacteria)

The entire nucleotide sequence of Escherichia coli genomic DNA is knownto the public (GenBanak accession number: U00096). Also, the amino acidsequence of Escherichia coli serine hydroxymethyltransferase and thenucleotide sequence (GenBank accession number: J01620) of the genethereof (hereafter to be abbreviated in some cases as glyA) have alreadybeen reported. PCR was carried out using genomic DNA of Escherichia colicell line W3110 (ATCC27325) as a template and oligonucleotides havingthe nucleotide sequences set forth in SEQ ID NOS: 15, 16, 17, and 18,which were produced based on genetic information regarding a region inthe vicinity of glyA of genomic DNA of Escherichia coli cell line W3110.The obtained DNA fragments were digested with BamHI and PstI and withPstI and HindIII, respectively, which are restriction enzymes. Thus,approximately 850 by and 750 by fragments of each DNA fragment wereobtained. The resulting DNA fragments were mixed with fragments obtainedby digesting a temperature-sensitive cloning vector pTH18cs1 (GenBankaccession number: AB019610) (Hashimoto-Gotoh, T., Gene, 241, 185-191(2000)) with BamHI and HindIII, followed by ligation using a ligase. Theresultant was transformed into a DH5α cell line at 30° C. Thus, atransformant that was able to grow on an LB agar plate containing 10μg/ml of chloramphenicol was obtained. The obtained colony was culturedovernight at 30° C. in an LB liquid medium containing 10 μg/ml ofchloramphenicol so that a plasmid was recovered from the obtained cellbodies. The recovered plasmid was digested with PstI so as to besubjected to blunt-end treatment with T4DNA polymerase. Thereafter, theplasmid was ligated with a tetracycline-resistant gene derived fromtransposon Tn10.

The thus obtained plasmid was transformed into Escherichia coliW3110dsdA-deficient cell line at 30° C., followed by overnight cultureat 30° C. on an LB agar plate containing 10 μg/ml of chloramphenicol and50 μg/ml of tetracycline. Thus, a transformant was obtained. An LBliquid medium containing 50 μg/ml of tetracycline was inoculated withthe obtained transformant, followed by overnight culture at 30° C. Next,the resultant was applied to an LB agar plate containing 50 μg/ml oftetracycline so as to obtain the culture cell bodies. Thus, coloniesthat grow at 42° C. were obtained. The obtained colonies were culturedovernight in an LB liquid medium containing 50 μg/ml of tetracycline at30° C. The resultant was further applied to an LB agar plate containing50 μg/ml of tetracycline so as to obtain colonies that grow at 42° C.

100 colonies were randomly picked up from the colonies that appeared.Each of them was allowed to grow on an LB agar plate containing 50 μg/mlof tetracycline and on an LB agar plate containing 10 μg/ml ofchloramphenicol. Then, chloramphenicol-sensitive clones that exclusivelygrow on an LB agar plate containing tetracycline were selected. Further,fragments (of approximately 3.6 kbp) in the region in the vicinity ofglyA were amplified by PCR from chromosome DNAs of these clones ofinterest. Then, a cell line in which glyA had been substituted with atetracycline-resistant gene was selected. The obtained cell line wasdesignated as a W3110dsdA/glyA-deficient cell line.

[13-2] (Effects of Escherichia Coli in which the GlyA Gene is Destroyed)

This Escherichia coli was transformed with plasmid pXDTA1, followed byculture in the same manner as that used in Example 10. Note that 20 mg/Lof glycine was added in the case of flask culture and 2 g/L of glycinewas added in the case of culture in a fermenter. During culture, glycineused was added at several different times.

L-serine production activity was examined in the same manner as thatused in Example 9. D-serine optical purity was 97%.

EXAMPLE 14 (Effects of Metal Salts)

300 mM EDTA (3.5 g; pH 7.5) was added to 100 g of frozen cell bodies ofMT-11028 obtained in Example 10, followed by agitation at 4° C. for 1hour. The resulting suspension (10 g) was suspended in 10 g of 0.5 Mpotassium phosphate buffer (pH 7.0) containing 20 mM each of manganesechloride, zinc sulfate, cobalt chloride, nickel chloride, calciumchloride, and ferrous chloride, followed by agitation at 4° C. for 1hour.

Next, formaldehyde was added to the above suspension so as to accountfor 0.5% of the resultant, followed by agitation at 35° C. for 1 hour.Cell bodies (weighing 0.22 g as dry cell bodies) were taken from theabove processed cell solution. The solution (9 g) used for enzymeactivity measurement described in Example 10 was added thereto, followedby agitation at 35° C. for 20 hours. Then, the ratio between L-serineproduced and residual D-serine was measured.

In a case in which a metal salt was used, optical purity of D-serine was96% or more. In addition, 50% or more of the activity of the enzymesynthesizing D-serine from formaldehyde and glycine was maintainedcompared with the activity before organic solvent treatment, even in acase in which a metal salt was used.

EXAMPLE 15 Method for Improving Optical Purity UsingL-Serine-Deaminase-Expressing Escherichia Coli

An LB medium (50 ml) was inoculated with Escherichia coli cell lineK-12. After overnight culture at 30° C., harvest was carried out,followed by bacteriolysis using a lytic solution containing lysozyme (1mg/ml). The resulting lysate was subjected to phenol treatment. Then,DNA was allowed to precipitate by ethanol precipitation in accordancewith a usual method. The resulting DNA precipitate was recovered byspooling it onto a glass rod and washed so as to be used for PCR.

Primers used for PCR were oligonucleotides (obtained by custom synthesisfrom Hokkaido System Science Co., Ltd.) having nucleotide sequences setforth in SEQ ID NOS: 19 and 20, respectively, which were designed basedon the known L-serine deaminase gene of Escherichia coli (GenBanakaccession number: M28695). These primers had EcoRI and HindIIIrestriction enzyme recognition sequences near the 5′ and 3′ ends,respectively.

With the use of 0.025 ml of a PCR reaction solution containing 6 ng/μleach of chromosome DNAs of the aforementioned microorganisms and 3 μMeach of the primers, PCR was carried out under the following conditions:denaturation at 96° C. for 1 minute, annealing at 55° C. for 30 seconds,and elongation reaction at 68° C. for 1 minute and 30 seconds for 35reaction cycles.

The PCR reaction product and plasmid pUC18 (Takara Shuzo) were digestedwith EcoRI and HindIII, followed by ligation using Ligation High(TOYOBO). Thereafter, the obtained recombinant plasmid was used fortransformation of Escherichia coli DH5α. The transformed cell line wascultured in an LB agar medium containing 50 μg/ml of ampicillin (Am) andX-Gal (5-bromo-4-chloro-3-indolyl-β-D-galactoside). Thus, anAm-resistant transformed cell line that was formed into a white colonywas obtained. A plasmid was extracted from the thus obtained transformedcell line. In accordance with a usual method for base sequencing, thenucleotide sequence of the DNA fragment that had been introduced intothe plasmid was confirmed to be identical to the sequence of a knownEscherichia coli L-serine deaminase. The obtained expression plasmid wasdesignated as pSDA1.

The Escherichia coli W3110dsdA/glyA-deficient cell line was transformedby a usual method using pSDA1. The obtained transformant was cultured ina fermenter in the same manner as that used in Example 13.

In the same manner as that used for the as comparative example inExample 12, reaction was carried out by adding 10 g of theaforementioned cell bodies to a reaction solution. The reaction solutionwas analyzed by HPLC. Thus, optical purity of D-serine in the reactionsolution was found to be 99.9%.

All publications, patents, and patent applications cited herein areincorporated herein by reference in their entirety.

INDUSTRIAL APPLICABILITY

The present invention is useful as a method for producing D-serine fromglycine and formaldehyde. In addition, D-serine obtained by theproduction method of the present invention is useful, for example, as amedicine intermediate of a starting material of D-cycloserine that isuseful as an antituberculous agent.

1-10. (canceled)
 11. A method for producing D-serine, wherein D-serineis synthesized by allowing glycine to react with formaldehyde in thepresence of a microorganism having activity of synthesizing D-serinefrom glycine and formaldehyde or a treated product thereof, the methodcomprising one or more of the following (i) to (iv) whereby formation ofL-serine as a byproduct in a reaction solution is restrained such thatL-serine accounts for 1.5 mol % or less relative to D-serine during thereaction: (i) allowing a microorganism having activity of synthesizingD-serine from glycine and formaldehyde to be subjected to an organicsolvent treatment and/or heat treatment; (ii) controlling formaldehydeconcentration in a reaction solution a) to 2M or less in a case in whichan organic solvent treatment and/or heat treatment are/is carried out bythe method (i) above and b) to from 150 mM to 2M inclusive in a case inwhich an organic solvent treatment and/or heat treatment are/is notcarried out; (iii) using, as a catalyst, a microorganism comprising anenzyme having activity of synthesizing D-serine from glycine andformaldehyde and lacking a L-serine synthase gene; and (iv) adding amicroorganism comprising an enzyme having an L-serine deaminase activityto a reaction solution.
 12. The production method according to claim 11,wherein the organic solvent is at least one selected from the groupconsisting of formaldehyde, benzaldehyde, dichloroethane, and isopropylalcohol.
 13. The production method according to claim 11 or claim 12,wherein the organic solvent treatment and/or heat treatment are/iscarried out in the presence of divalent metal ions.
 14. The productionmethod according to claim 13, wherein the divalent metals are one ormore types of metals selected from the group consisting of magnesium,manganese, zinc, nickel, cobalt, and iron.
 15. The production methodaccording to claim 11, wherein the microorganism having activity ofsynthesizing D-serine from glycine and formaldehyde is a transformantobtained by transforming a host cell, which is optionally aD-serine-deaminase-deficient microorganism, using recombinant DNAconstructed by integrating DNA encoding a protein comprising the aminoacid sequence of SEQ ID NO: 4, 6, or 8 or a protein comprising an aminoacid sequence derived from the amino acid sequence amino acid sequenceset forth in SEQ ID NO: 4, 6, or 8 by deletion, substitution, insertion,or addition of one or more amino acid residues and having enzymeactivity of synthesizing D-serine from glycine and formaldehyde.
 16. Theproduction method according to claim 11, wherein the microorganism hasactivity of synthesizing D-serine from glycine and formaldehyde in areaction solution having a high formaldehyde concentration of 410 mM.17. The production method according to claim 11, wherein themicroorganism is a transformant obtained by transforming a host cellusing a recombinant DNA constructed by integrating, into a vector, DNAencoding a protein described in the following (a) or (b): a) D-threoninealdolase from Xanthomonas oryzae or b) a protein comprising an aminoacid sequence derived from the amino acid sequence of (a) by deletion,substitution, insertion, or addition of 1 to 5 amino acid residues andhaving enzyme activity of synthesizing D-serine from glycine andformaldehyde.
 18. The production method according to claim 11, whereinthe microorganism is a transformant obtained by transforming a host cellusing a recombinant DNA constructed by integrating, into a vector, DNAencoding a protein comprising the consensus sequence of the followingfour sequences: (a) SEQ ID NO:4; (b) SEQ ID NO:6; (c) SEQ ID NO:8, and(d) an amino acid sequence of D-threonine aldolase from Xanthomonasoryzae described in GenBank accession number E05055, and the protein hasenzyme activity of synthesizing D-serine from glycine and formaldehyde.